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PCR-RFLP primer for distinguishing DHV-1 and new serotype and method

A technology of PCR-RFLP and duck hepatitis virus, which is applied in the field of PCR-RFLP primers for distinguishing duck hepatitis virus type 1 and new serotypes, can solve the problems of cumbersome optimization of double PCR conditions, and achieve high speed, high accuracy and sensitivity high effect

Inactive Publication Date: 2015-12-09
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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Problems solved by technology

However, related methods need to design specific primers for duck hepatitis virus type 1 and new serotypes respectively, and establish double PCR detection, but the optimization of double PCR conditions is cumbersome.

Method used

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  • PCR-RFLP primer for distinguishing DHV-1 and new serotype and method
  • PCR-RFLP primer for distinguishing DHV-1 and new serotype and method

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Embodiment 1

[0028] 1. Virus strains:

[0029] Duck hepatitis virus type 1 and new serotypes were preserved by the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences.

[0030] 2. Primer design and synthesis

[0031] The primers P1 and P2 were designed according to the gene characteristics of duck hepatitis virus type 1 and the new serotype. The sequences of primers P1 and P2 were: upstream primer P1: 5'-CAATTGAGGACATGGCTAAGAAA-3', downstream primer P2: 5'-TCAGAGASCCRTCRAAWCCAGAAA-3'.

[0032] 3. RT-PCR amplification

[0033] The nucleic acid RNA of duck hepatitis virus type 1 and new serotypes was extracted by conventional methods. The designed specific primers P1 and P2 were used for RT-PCR amplification, and the size of the amplified fragment was about 555bp. RT-PCR amplification (take Treasure Bioengineering (Dalian) Co., Ltd. PrimeScript? OneStepRT-PCRKitVer.2 (DyePlus) as an example) system is 50μL, including PrimeScript1StepEnzymeMix2μL,...

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Abstract

The invention discloses a PCR-RFLP primer for distinguishing DHV-1 and a new serotype and a method for distinguishing the DHV-1 and the new serotype. The primer is an upstream primer P1:5'-CAATTGAGGACATGGCTAAGAAA-3' and a downstream primer P2:5'-TCAGAGASCCRTCRAAWCCAGAAA-3. According to the method, distinguishing is conducted according to the DHV-1 and the restriction enzyme cutting site difference contained in a new serotype coding area and includes the steps that RNA extraction is conducted, and after corresponding target fragments are obtained through RT-PCR amplification, RFLP analysis is conducted after XhoI restriction enzyme cutting. The distinguishing method is simple and fast in operation and high in accuracy.

Description

technical field [0001] The invention belongs to the technical field of rapid diagnosis of veterinary infectious diseases, and in particular relates to a PCR-RFLP primer for distinguishing duck hepatitis virus type 1 from a new serotype and a method thereof. Background technique [0002] Duck hepatitis virus (Duckhepatitis virus, DHV) is an acute, highly contagious infectious disease caused by duck hepatitis virus, mainly affecting ducklings under 3 weeks old, with hepatitis as the typical feature. The disease has an acute onset, short course, high morbidity and mortality, and seriously threatens the healthy development of the duck industry [Yang Weixing, Shi Shaohua, Chen Hongmei, et al. Sequence analysis of three strains of duck hepatitis virus type 1 genome[J]. China Agricultural Science Bulletin, 2010, 26 (14): 8-12. ]. [0003] In 1999, Su Jingliang of China Agricultural University isolated two new serotypes of duck hepatitis virus (Duckhepatitis virus newserotype, DH...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/706C12Q2521/301C12Q2531/113
Inventor 万春和黄瑜施少华傅光华陈红梅程龙飞傅秋玲陈珍陈翠腾
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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